Method for diagnosing and treating dentinogenesis imperfecta type II by using dentin sialophsphoprotein (dspp)gene and its encoding product

ABSTRACT

The invention has disclosed a method for diagnosis of dentinogenesis imperfecta type II (DGI-II) and/or dentinogenesis imperfecta type II with deafness (DGI-II with deafness). Said method comprises the steps of detecting the DSPP gene, transcript and/or protein in said subject and comparing it with the normal DSPP gene, transcript and/or protein to determine whether there is any variation, wherein said variation indicates that the possibility of suffering DGI-II and/or DGI-II with deafness in said subject is higher than the normal population. The present invention also discloses the method and pharmaceutical composition for treating DGI-II and/or DGI-II with deafness.

FIELD OF INVENTION

[0001] This invention relates to both biological engineering and medicalfields. In particular, it relates to a method of diagnosing and treatingdentinogenesis imperfecta type II using human dentin sialophosphoproteinor DSPP gene and the coded product, and a pharmaceutical compositioncontaining DSPP gene and/or protein.

TECHNICAL BACKGROUND

[0002] The odontoblasts produce the dentin, which consists in maturetooth or the tooth during tooth development phase. Duringdentinogenesis, the odontoblasts form dentinal tubules. Dentin cellprocesses in these tubules make dentin a living tissue. During theprimary stage of dentinogenesis, the odontoblasts synthesize, secreteand reabsorb the dentin matrix components. Protein synthesis occurswithin cells. Exocytosis and endocytosis occurs mainly in cellprocesses. The first material formed is unmineralized mantle dentinmatrix, mainly including collagen secreted by cells and non-collagenouscomponents. The fasciculata collagen fibers congregate to a ballstructure. Due to the continual increase of new fibrils, collagenbecomes closer and closer. As a result, these prophase collagen fiberschange into collagen fibers. Thus predentin characterized by collagenmatrix is formed. Later, the mineralization crystals gradually depositedto become dentin at some distance away from cells.

[0003] The mature dentin contains more inorganic minerals than the bone.65 wt. % of dentin are minerals, mostly hydroxyapatite crystals. Organicmaterials are 20%, mainly collagenous proteins and non-collagenousproteins. These collagens offer braces to the deposition ofhydroxyapatite plate like crystalline.

[0004] Type I collagen is predominant (about 97%) in dentin collagens,10%-15% of which is type I collagen trimer. Different from otherconnective tissue, type III collagen is lacking in dentin. Moreover,there are types V and VI collagens in dentin, but the contents aresmall. Although the contents of non-collagenous proteins in dentin aresmall, there are various kinds. According to the source of proteins, thedentin noncollagenous proteins can be divided to four kinds: dentinspecific protein, mineralized tissue specific protein, aspecificprotein, and blood serum source protein (or dentin affinity protein).Dentin specific protein is the only one which is synthesized andsecreted by odontoblasts and exists only in dentin. Mineralized tissuespecific proteins means those that are found and exist not only indentin but also in cementum and bone. They are synthesized and secretedby osteoblasts, odontoblasts and cementoblasts. The non-specificproteins exist both in dentin and other tissues, including parenchyma,and synthesized and secreted by odontoblasts and other kinds of cells.Blood serum source proteins are those which are synthesized by othercells in the body, mainly by liver cells, and secreted to serum. Theseproteins have a high affinity to dentin, though they are not synthesizedby dentin. They can enter dentin by blood circulation, so they are alsocalled dentin affinity proteins. Proteoglycans or PGS are other primarynon-collagenous proteins in dentin. They are large covalent moleculesformed by many anylose side chains and one core protein. These sidechains are composed of repeating disaccharide chain units, each of whichconsists of one glycuronic acid and one Nacetamidoacetose. One functionof PGS in dentinogenesis is to affect or even control the systematism ofcollagen skeleton in predentin. The dentin proteoglycans fixed on thesolid bracket can induce the formation of hydroxyapatite in vivo and inphysiologic pH and ionic condition in vitro. On the contrary, the liquidproteoglycans restrain the form of mineral components in vitro. Thecombination of PGS and Ca²⁺ is the precondition of inducing theformation of hydroxyapatite.

[0005] Dentinogenesis imperfecta or DGI is an autosomal dominant dentalgenetic disease that has a prevalence of {fraction (1/8000)}. There arethree types according to clinical taxonomy⁽¹⁾ (The number in bracketsshows the relative literature.). Dentinogenesis imperfecta Type I isalso named DGI-I. Except for dentinogenesis imperfecta, patients usuallyhave osteogenesis imperfecta The pathogeny is broad mutations incollagen type I gene⁽²⁾. Type II or DGI-II is also called hereditaryopalescent dentin. DGI-II has a relationship with the impropermineralization of dentin and its penetrance is nearly 100%⁽³⁾. Type IIIor DGI-III is also called dentinogenesis imperfecta Brandywine type orisolate hereditary opalescent dentin. It is a special hereditaryopalescent dentin, only found in three isolates in Washington, D.C., theState of Maryland, USA. Witkop first reported this illness in 1956⁽⁴⁾and there is no related report in China till now. DGI-III has anobviously genetic heterogeneity. Its pathogeny is related tomalamineralization. Because the gene causing DGI-I has been found andDGI-III is only found in the isolates in the State of Maryland, USA,DGI-II becomes the focus of tooth endodontics.

[0006] The clinical symptoms and pathology changes of DGI-II are asfollows. The malajustment and turbulence of mineralization result inembryonic layer dysplasia in dentin. Both the primary dentition andpermanent dentition are affected, with a more serious damage in primarydentition. A predominant feature is a blue-gray or amber browndiscoloration of the teeth. The improper mineralized dentin is soft andthe crown is prone to be worn. Moreover, compensatory hyperplasia ofmatrix increases in improperly mineralized dentin, leading to small orobliterated pulp chambers. Radiographs reveal that the affected teethhave bulbous crowns, narrow roots and small or obliterated pulp chambersand root canals. The pathology shows that the enamel surface is normal,but hypoplasia and hypocalcification can be found in about ⅓ of thepatients. The enamel dentin junction changes greatly. Some teeth have anon-obvious sector structure in the enamel dentin junction. However,others are especially obvious. Dentin is lamellar with nearly normalouterdentin and dentinal tubules having subdivisional branches. In otherparts, the dentin is obviously abnormal. Some short tubules or tubuleswith abnormal form distribute in dentin matrix disorderly. The predentinzone is very wide. Along the plywood, the remaining embedded cell can beseen, similar to embedded odontoblast and bloods. Observation underelectron microscope indicates that the form and size of hypoplasticdentin micro-crystal are unchanged, but the quantity is small.Uncalcified or partly calcified transverse collagen fasciculi andvolumes of crystal space can be seen discontinuously.

[0007] For the mapping of DGI-II gene, in 1969, Bixler et al.(⁵) triedto use some protein polymorphic markers, such as ABO, Rh, MNSs, Ke11,Fy, JK, HP, ACP1, PGM1 and PTC, to perform a linkage analysis in DGI-IIfamilies, but they failed to get the linkage evidence. In 1977,Mikkelsen et al.⁽⁶⁾ mapped a group of specific components (GC) inVitamin D conjugated protein to 4q11-q13. In the next year, Kühnlidentified that GC included six phenotypes: GC2/2, 2/1+, 2/1−, 1+/1−,1+/1+, and 1−/1−. Later, Ball. S. P. et al.⁽⁷⁾ analyzed the linkage in aDGI-II big family named Family MRC4000 with the polymorphic markers ofGC and found that DGI-II had a close linkage with GC (Lod=+7.9, θ=0.13).In 1992, Crall et al.⁽⁸⁾ mapped DGI-II to interval defined by twoprotein polymorphic markers: GC and interferon-inducible cytokineINP-10. The relative chromosome location was 4q12-21. The results aboveonly offered a gross orientation of disease gene of DGI-II. Under thatcondition, it was almost impossible to clone the disease gene in thisregion.

[0008] In 1995, Crosby A.H et al⁽⁹⁾ analyzed the linkage in two bigDGI-II families with 9 short tandem repeat polymorphic markers (STRP)and mapped the disease gene to the 4q21-23 region defined by two STRPsof D4S2691 and D4S2692. Multipoints linkage analysis suggested that thedisease gene might be in the region within about 3.2 cM around SPP1.Recently, Aplin H. M et al.⁽¹⁰⁾ genotyped two big families used byCrosby A. H with 5 hyperdense STRPs. The linkage analysis showed thatthe disease gene of DGIII located between two STRPs of GATA62A11 andD4S1563 with a genetic distance of 2 cM. Moreover, this research groupestablished the YACs Contigs in this region. They also identified thatDMP1, IBSP, SPP1 and DSPP are all in this candidate region by PCRtechnology.

[0009] However, the mechanism of dentinogenesis imperfecta type II isstill unclear so far. Also the direct relationship betweendentinogenesis imperfecta type II and some special kind of protein isnot reported.

[0010] In addition, there is still no effective method to diagnoseDGI-II early and/or antenatally and to cure DGI-II by non-operativetreatment in the art.

[0011] Therefore, there is an urgent need to develop new and efficientmethods to diagnose and cure DGI-II, the relative pharmaceuticals, anddiagnostic technology and reagents.

SUMMARY OF INVENTION

[0012] One purpose of the invention is to provide a new diagnosticmethod and detection kit, especially for antenatal and/or earlydiagnosis of dentinogenesis imperfecta type II (DGI-II) anddentinogenesis imperfecta type II with deafness (DGI-II with deafness).

[0013] Another purpose is to provide a new method to treat DGI-II andDGI-II with deafness.

[0014] Still another purpose is to provide a pharmaceutical compositionto treat DGI-II and DGI-II with deafness.

[0015] In the first aspect, the invention provides a method fordetermining the susceptibility of DGI-II and/or DGI-II with deafness ina subject comprising the steps of:

[0016] detecting the DSPP gene, transcript and/or protein in saidsubject and comparing it with the normal DSPP gene, transcript and/orprotein to determine whether there is any difference,

[0017] wherein said difference indicates that the possibility ofsuffering DGI-II and/or DGI-II with deafness in said subject is higherthan the normal population.

[0018] In a preferred embodiment, the DSPP gene or transcript isdetected, and compared with the normal DSPP nucleotide sequence todetermine the difference. More preferably, said difference is selectedfrom the group consisting of: in position 1 of Exon 3, G1→T1; inposition 1 of Intron 3, G1→A1.

[0019] In the second aspect, the invention provides a method fortreating DGI-II and/or DGI-II with deafness comprising the step ofadministrating a safe and effective amount of normal DSPP and/or DSPprotein to the patient in need of said treatment. Preferably, the DSPPand/or DSP protein are administrated topically to periodontal tissues.

[0020] In the third aspect, the invention provides a pharmaceuticalcomposition comprising a safe and effective amount of DSPP and/or DSPprotein and a pharmaceutically acceptable carrier. Preferably, saidpharmaceutical composition is injection.

[0021] In the fourth aspect, the invention provides a kit for detectingDGI-II and/or DGI-II with deafness comprising the primers whichspecifically amplify the DSPP gene or transcript. Preferably, the kitfurther comprises a probe that binds to the site of mutation.

[0022] In view of the technical teaching of the invention, the otheraspects of the invention will be apparent to the skilled in the art.

DESCRIPTION OF DRAWINGS

[0023]FIG. 1 shows the gene structure of DSPP. This gene contains 5exons and 4 introns. The full length is 8210 bp. Exon 1 (7-98), Exon 2(2359-2437), Exon 3 (3577-3660), Exon 4 (3794-4780) and Exon 5(5257-8201) encode DSPP. Exons 1-4 and part of Exon 5 (5257-5520) encodeDSP, while another part of Exon 5 (5521-7893) encodes DPP.

[0024]FIG. 2 shows the haplotype construction of STRP markers in 4q21region in a dentinogenesis imperfecta type II family.

[0025]FIG. 3 shows the haplotype construction of STRP markers in 4q21region in a DGI-II with deafness family.

[0026]FIGS. 4A and 4B show mutations in DSPP gene. FIG. 4A shows G1→T1in position 1 of Exon 3, which causes codon GTT change into TTT,resulting in a corresponding amine acid change of Val→Phe. FIG. 4B showsG1→A1 in position 1 of Intron 3, which causes the mutation of splicingsite.

DETAILED DESCRIPTION OF INVENTION

[0027] After studying for several years, the inventors of the inventionhave, for the first time found and proved dentin sialophosphoprotein(DSPP) and/or dentin sialoprotein (DSP) have a close relationship withdentinogenesis imperfecta type II (DGI-II). In addition, the newfunction of DSPP/DSP was found, i.e., the changes of DSPP or DSP willcause DGI-II directly. Based on this discovery, the inventorsaccomplished the invention.

[0028] Firstly, the inventors collected two genetic families affected bydentinogenesis imperfecta or dentinogenesis imperfecta with progressivehearing loss in China. Then they localized the disease gene ofdentinogenesis imperfecta to the 4q21-22 region in Chromosome 4 bygenotyping and linkage analysis with microsatellite markers Then, theinventors identified the candidate genes by the following steps:

[0029] (1) Finding all of the genes mapped in 4q21-22 region, i.e.,making the transcription map in 4q21-22 region;

[0030] (2) Checking the expression situation of all of the genes in4q21-22 region;

[0031] (3) Determining the genes mapped in 4q21-22 region and expressedin dental pulp as the candidates for dentinogenesis imperfecta.

[0032] The results showed that the candidate genes included DMP1, IBSP,SPP1, DSP, DPP and DSPP.

[0033] Further, the inventors used PCR-SSCP technique to screen allcandidate genes for mutation and found that the mutations in DSPP have adirect causality with dentinogenesis imperfecta, while other genes donot.

[0034] Finally, the mode and site of DSPP mutation in two geneticfamilies were identified by sequence analysis. In DGI-II family,sequencing revealed a G1→T1 mutation at position 1 of Exon 3 (position3577 in SEQ ID NO:1). The mutation results in not only an amino acidchange, but also a splicing site change which may cause the expressionof intron, termination of translation in advance or frame shifting (FIG.4A). Therefore, the normal DSPP (or DSP) protein is unable to beexpressed.

[0035] In another DGI-II with deafness family, the mutation is a G1→A1mutation in position 1 of Intron 3 (position 3661 of SEQ ID NO:1). Themutation was predicted to result in splicing site change, which maycause the expression of intron, termination of translation in advance orframe shifting (FIG. 4B). Therefore, the normal DSPP (or DSP) protein isunable to be expressed. Further, it may influence the translation ofsignal peptide so that DSPP can not be correctly localized.Surprisingly, this mutation causes the patient affected with both DGI-IIand deafness, suggesting that DSPP mutation is associated with deafness.It is possible to diagnose deafness, especially DGI-II with deafness, bydetecting whether DSPP is normal or not.

[0036] On the basis of this invention, one can design and exploit newdrugs based on DSPP gene and its products (e.g., transcripts andproteins) as well as the interacting molecule. In addition, one can useDSPP gene in vitro to reconstruct teeth or remodel some tooth structure,such as dentin.

[0037] Human DSPP mutation causes human dentinogenesis imperfecta typeII. Based on the DSPP gene and its expression products, one can developnew drugs and diagnosis/treatment techniques for detecting and treatinghuman DGI-II.

[0038] Human DSPP Gene and Protein

[0039] The detailed sequences of human DSPP gene and protein areavailable in Genbank (The accession number is AF163151) and somereferences, such as Gu, K., Chang, S., Ritchie, H. H., Clarkson, B. H.and Rutherford, R. B., Eur. J. Oral Sci. 2000 Feb: 108 (1):35-42. InSequence Listing, human DSPP nucleotide sequence and amino acid sequenceare shown in SEQ ID NO:1 and SEQ ID NO: 2, respectively. FIG. 1 showsthe introns and exons of human DSPP. Exon 1 7-98 mRNA join (7-98,2359-2437, 3577-3660, 3794-4780, 5257-8201) Exon 2 2359-2437 CDS join(2387-2437, 3577-3660, 3794-4780, 5257-7896) sig_peptide 2387-2431mat_peptide join (2432-2437, 3577-3660, 3794-4780, 5257-7893)/ Product“DSPP” mat_peptide join (2432-2437, 3577-3660, 3794-4780, 5257-5520)/Product “DSP” Exon 3 3577-3660 Exon 4 3794-4780 Exon 5 5257-8201mat_peptide 5521-7893 misc_feature 5596-5604 /note = “Region: cellbinding domain” PolyA_signal 7988-7993 PolyA_signal 8171-8176

[0040] The DSPP and/or DSP protein or polypeptide have various usesincluding but not limited to: curing disorders caused by low or noactivity of DSPP and/or DSP protein (using directly as a medicine), andscreening out antibodies, polypeptides or ligands which promote thefunction of DSPP and/or DSP. The expressed recombinant DSPP and/or DSPprotein can be used to screen polypeptide library to findtherapeutically valuable polypeptide molecules which activate thefunction of DSPP and/or DSP protein.

[0041] In another aspect, the invention also includes polyclonal andmonoclonal antibodies, preferably monoclonal antibodies, which arespecific for polypeptides encoded by human DSPP DNA or fragmentsthereof. By “specificity”, it is meant an antibody that binds to thehuman DSPP gene products or fragments thereof. Preferably, the antibodybinds to the human DSPP gene products or fragments thereof and does notsubstantially recognize nor bind to other antigenically unrelatedmolecules. Antibodies that bind to human DSPP and block human DSPPprotein and those which do not affect the human DSPP function areincluded in the invention.

[0042] The present invention includes not only intact monoclonal orpolyclonal antibodies, but also immunologically-active antibodyfragments, e.g., a Fab′ or (Fab)₂ fragment, an antibody heavy chain, anantibody light chain, a genetically engineered single chain Fv molecule(Lander, et al., U.S. Pat. No. 4,946,778), or a chimeric antibody, e.g.,an antibody which contains the binding specificity of a murine antibody,but the remaining portion of which is of human origin.

[0043] The antibodies in the present invention can be prepared byvarious techniques known to those skilled in the art. For example,purified human DSPP gene products, or its antigenic fragments can beadministrated to animals to induce the production of polyclonalantibodies. Similarly, cells expressing human DSPP or its antigenicfragments can be used to immunize animals to produce antibodies. Theantibodies of the invention can be monoclonal antibodies which can beprepared by using hybridoma technique (See Kohler, et al., Nature, 256;495,1975; Kohler, et al., Eur. J. Immunol. 6: 511,1976; Kohler, et al.,Eur. J. Immunol 6: 292, 1976; Hammerling, et al., In MonoclonalAntibodies and T Cell Hybridomas, Elsevier, N.Y., 1981). Antibodies ofthe invention comprise those which block human DSPP function and thosewhich do not affect human DSPP function. Antibodies in the invention canbe produced by routine immunology techniques and using fragments orfunctional regions of human DSPP gene product. These fragments andfunctional regions can be prepared by recombinant methods or synthesizedby a polypeptide synthesizer. The antibodies binding to unmodified humanDSPP gene product can be produced by immunizing animals with geneproducts produced by prokaryotic cells (e.g., E. coli), and theantibodies binding to post translationally modified forms thereof (e.g.,glycosylated or phosphorylated polypeptide) can be acquired byimmunizing animals with gene products produced by eukaryotic cells(e.g., yeast or insect cells).

[0044] The antibody against human DSPP and/or DSP protein can be used inimmunohistochemical method to detect the presence of DSPP and/or DSPprotein in the biopsy specimen.

[0045] The polyclonal antibodies can be prepared by immunizing animals,such as rabbit, mouse, and rat, with human DSPP and/or DSP protein.Various adjuvants, e.g., Freund's adjuvant, can be used to enhance theimmunization.

[0046] The substances that act with DSPP and/or DSP protein, e.g.,inhibitors, agonists and antagonists, can be screened out by variousconventional techniques, using the protein of the invention.

[0047] The protein, antibody, inhibitor, agonist or antagonist of theinvention provides different effects when administrated in therapy.Usually, these substances are formulated with a non-toxic, inert andpharmaceutically acceptable aqueous carrier. The pH typically rangesfrom 5 to 8, preferably from about 6 to 8, although pH may alteraccording to the property of the formulated substances and the diseasesto be treated. The formulated pharmaceutical composition isadministrated in conventional routine including, but not limited to,intramuscular, intravenous, subcutaneous, or topical administration. Thetopical administration at periodontal tissues is preferred.

[0048] The normal DSPP and/or DSP can be directly used for curingdisorders, e.g., DGI-II. The DSPP and/or DSP protein of the inventioncan be administrated in combination with other medicaments for DGI-II.

[0049] The invention also provides a pharmaceutical compositioncomprising safe and effective amount of DSPP and/or DSP protein incombination with a suitable pharmaceutical carrier. Such a carrierincludes but is not limited to saline, buffer solution, glucose, water,glycerin, ethanol, or the combination thereof. The pharmaceuticalformulation should be suitable for the delivery method. Thepharmaceutical composition of the invention may be in the form ofinjections which are made by conventional methods, using physiologicalsaline or other aqueous solution containing glucose or auxiliarysubstances. The pharmaceutical compositions in the form of tablet orcapsule may be prepared by routine methods. The pharmaceuticalcompositions, e.g., injections, solutions, tablets, and capsules, shouldbe manufactured under sterile conditions. The active ingredient isadministrated in therapeutically effective amount, e.g., from about lugto 5 mg per kg body weight per day. Moreover, the polypeptide of theinvention can be administrated together with other therapeutic agents.

[0050] When using pharmaceutical composition, the safe and effectiveamount of the DSPP and/or DSP protein or its antagonist or agonist isadministrated to mammals. Typically, the safe and effective amount is atleast about 0.1 ug/kg body weight and less than about 10 mg/kg bodyweight in most cases, and preferably about 0.1-100 ug/kg body weight. Ofcourse, the precise amount will depend upon various factors, such asdelivery methods, the subject health, and the like, and is within thejudgment of the skilled clinician.

[0051] The human DSPP and/or DSP polynucleotides also have manytherapeutic applications. Gene therapy technology can be used in thetherapy of abnormal cell proliferation, development or metabolism, whichis caused by the loss of DSPP and/or DSP expression or the expression ofabnormal or non-active DSPP and/or DSP. The methods for constructing arecombinant virus vector harboring DSPP and/or DSP gene are described inthe literature (Sambrook, et al.). In addition, the recombinant DSPPand/or DSP gene can be packed into liposome and then transferred intothe cells.

[0052] The methods for introducing the polynucleotides into tissues orcells include: directly injecting the polynucleotides into tissue in thebody, in vitro introducing the polynucleotides into cells with vectors,such as virus, phage, or plasmid, and then transplanting the cells intothe body.

[0053] The invention further provides diagnostic assays for quantitativeand in situ measurement of DSPP and/or DSP protein level. These assaysare well known in the art and include FISH assay and radioimmunoassay.The level of DSPP and/or DSP protein detected in the assay can be usedto illustrate the importance of DSPP and/or DSP protein in diseases andto determine the diseases associated with DSPP and/or DSP protein.

[0054] A method of detecting the presence of DSPP and/or DSP protein ina sample by utilizing the antibody specifically against DSPP and/or DSPprotein comprises the steps of: contacting the sample with the antibodyspecifically against DSPP and/or DSP protein; observing the formation ofantibody complex which indicates the presence of DSPP and/or DSP proteinin a sample.

[0055] The polynucleotide encoding DSPP and/or DSP protein can be usedin the diagnosis and treatment of DSPP and/or DSP protein relateddiseases. In respect of diagnosis, the polynucleotide encoding DSPPand/or DSP can be used to detect whether DSPP and/or DSP is expressed ornot, and whether the expression of DSPP and/or DSP is normal orabnormal, e.g., in the case of diseases. DSPP DNA sequences can be usedin the hybridization with biopsy samples to determine the expression ofDSPP. The hybridization methods include Southern blotting, Northernblotting and in situ blotting, etc., which are public and sophisticatedtechniques. The corresponding kits are commercially available. A part ofor all of the polynucleotides of the invention can be used as probe andfixed on a microarray or DNA chip for analyzing the differentialexpression of genes in tissues and for the diagnosis of genes. The DSPPand/or DSP specific primers can be used in RNA-polymerase chain reactionand in vitro amplification to detect the transcripts of DSPP and/or DSP.

[0056] Further, detection of the mutation of DSPP and/or DSP gene isuseful for the diagnosis of DSPP and/or DSP protein related diseases.The mutation forms of DSPP and/or DSP include site mutation,translocation, deletion, rearrangement and any other mutations comparedwith the normal wild-type DSPP and/or DSP DNA sequence. The conventionalmethods, such as Southern blotting, DNA sequencing, PCR and in situblotting, can be used to detect mutation. Moreover, mutation sometimesaffects the expression of protein. Therefore, Northern blotting andWestern blotting can be used to indirectly determine whether the gene ismutated or not.

[0057] The invention is further illustrated by the following examples.It is appreciated that these examples are only intended to illustratethe invention, but not to limit the scope of the invention. For theexperimental methods in the following examples, they are performed underroutine conditions, e.g., those described by Sambrook et al., inMolecule Clone: A Laboratory Manual, New York: Cold Spring HarborLaboratory Press, 1989, or as instructed by the manufacturers, unlessotherwise specified.

EXAMPLE 1

[0058] DGI-II family had 42 members, and DGI-II with deafness family had14 members. All individuals were subjected to careful clinicalexamination and recorded in details by experienced dentists. Thepatients with deafness were examined carefully by otologists andidentified by pure tone audiogram and brain stem evoked potential. 5 mlblood samples in the families were collected by standard venipunctureand stocked by ACD solution. DNA was extracted using the followingmethod:

[0059] Preparation of Blood DNA Sample

[0060] Blood DNA samples were extracted by Qiagen kit according tomanufacturer's instructions. The steps were as follows:

[0061] a. Add 20 ul Proteinase K, 200 ul blood sample and 200 ul BufferAL into a 1.5 ml microcentrifuge tube. Mix by pulse-vortexing for 15seconds.

[0062] b. Incubate for digestion at 56° C. for 10 minutes. Add 210 ul100% ethanol to the sample, and briefly centrifuge for 10 seconds.

[0063] c. Carefully apply the mixture onto a QIAamp spin column andcentrifuge at 8000 rpm for 1 minute.

[0064] d. Discard the filtrate and transfer the QIAamp spin column inanother 2 ml collection tube.

[0065] e. Add 500 ul Buffer AW1 into QIAamp spin column, centrifuge at8000 rpm for 1 minute.

[0066] f. Discard the filtrate and add 500 ul Buffer AW2 into QIAampspin column, centrifuge at 14000 rpm for 3 minutes.

[0067] g. Discard the filtrate and place the QIAamp spin column in a new1.5 ml microcentrifuge tube.

[0068] h. Add 200 ul Buffer AE into QIAamp spin column, incubate at roomtemperature for 5 minutes, and centrifuge at 8000 rpm for 1 minute. Thefiltrate collected in the tube was DNA solution from blood sample.

[0069] i. DNA quality was determined by 1% agarose gel electrophoresis.The DNA quantity was determined by UV spectrophotometer. The DNA sampleswere stored at −20° C.

EXAMPLE 2

[0070] 1 Genotyping:

[0071] The sequences of high polymorphic STR markers in region 4q21 wereobtained from Genome Database and markers A-G were D4S2691, D4S1534,GATA62 μl 1, DSP, DMP1, SPP1, D4S451, respectively. PCR amplificationswere carried out following LI-COR company manual for the touchdownprogram and using PTC-225 DNA Engine Tetrad (MJ-Research Inc.). PCRreactions were in 10 ul system containing 20 ng genomic DNA template,2.0 mM dNTP, 1.0 pmol M13-tailed forward primer and reverse primer, 1.0pmol fluorescent M13 primer, 1.5 mM MgCl₂, 10 mM Tris-HCl, and 1UAmpliTaq Gold Taq Polymerase (Perkin-Elmer Corp.). The reaction systemwas initially denatured at 95° C. for 8 minutes, followed by 4 cycles ofdenaturing at 95° C. for 45 seconds, annealing at 68° C. for 2 minuteswith a drop of 2° C. per cycle until 60° C., and extending at 72° C. for1 minute, and by a second set of 2-4 cycles of denaturing at 95° C. for45 seconds, annealing at 58° C. for 1 minute with a drop of 2° C. percycle until 50-54° C., and extending at 72° C. for 1 minute, and then by20-30 cycles of denaturing at 95° C. for 30 seconds, annealing at 50-54°C. for 30 seconds and extending at 72° C. for 30 seconds. Finally thesamples were extended at 72° C. for 15 minutes. PCR products andfluorescent-labeled standard size DNA markers were electrophoresed on aLI-COR automated sequencer on a polyacrylamide gel. Data were collectedand analyzed by Base Image 4.1 and Gene Image 3.12 software, whilelinkage ready pedigree files were generated. These files were used forlinkage analysis and haplotype analysis.

[0072] 2. Linkage Analysis and Haplotype Analysis

[0073] DGI-II hereditary locus was modeled as an autosomal dominantinheritance with 100% penetrance in a two-allele system. The frequencyof disease gene was set to 0.0001, the frequencies of STRs were assumedto be uniformly distributed. Two-point linkage analysis was performed byusing MLINK and ILINK program from the LINKAGE version 5.10 softwarepackage. Haplotype construction was performed using SIMWALK2 version2.31 and Cyrillic version 2.02 software.

[0074] The pedigree data are shown in Tables 1-2 and FIGS. 2-3. TABLE 1Disease locus in DGI-II pedigree and STRP two-point linkage analysis in4q21 region Loca- tion Lod score at θ Maximum marker 0.0 0.01 0.05 0.10.2 0.3 0.4 Lod θ A −∞ −0.11 2.19 2.76 2.59 1.81 0.83 2.76 0.1 B 1.651.62 1.51 1.37 1.09 0.78 0.42 1.65 0.0 C 7.63 7.50 6.96 6.25 4.74 3.091.36 7.63 0.0 D 6.06 5.96 5.53 4.98 3.82 2.57 1.24 6.06 0.0 E 8.24 8.117.54 6.80 5.22 3.49 1.67 8.24 0.0 F 8.38 8.24 7.67 6.93 5.32 3.55 1.648.38 0.0 G 7.34 7.23 6.77 6.16 4.87 3.44 1.84 7.34 0.0

[0075] TABLE 2 Disease locus in DGI-II with deafness pedigree and Lodscore in 4q21 region Loca- tion Lod score at θ Maximum marker 0.0 0.010.05 0.1 0.2 0.3 0.4 Lod θ A −∞ −2.86 −1.48 −0.92 −0.42 −0.18 −0.05−0.05 0.4 B −∞ 0.67 1.19 1.25 1.04 0.65 0.21 1.25 0.1 C 1.20 1.8 1.070.93 0.63 0.33 0.08 1.20 0.0 D −0.14 −0.09 −0.05 −0.02 −0.00 −0.00 −0.00−0.00 0.2 E 0.91 0.92 0.91 0.86 0.67 0.41 0.14 0.92 0.01 F 2.71 2.662.46 2.21 1.65 1.02 0.37 2.71 0.0 G 2.11 2.07 1.91 1.70 1.24 0.73 0.232.07 0.0

[0076] The results suggested that the disease genes in DGI-II and DGI-IIwith deafness pedigrees were linked with STRP markers in 4q21 region.

EXAMPLE 3

[0077] Mutation Screening of Candidate Genes

[0078] Using Primer 5.0 software (http://www/PrimerBiosoft.com), wedesigned primers to amplify exons and the splice junctions between exonsand introns of DSP gene (Table 3). PCR-SSCP technique was used to screenDSP gene for mutation. PCR products were electrophoresed on 10%polyacrylamide gel and 9.3% polyacrylamide gel with 4% glycerol. Then,the gels were silver stained according to standard protocol.

[0079] Primers were as follows: TABLE 3 Primer Sequences in DSPP CodingRegion Primer Name Sequence No. bp DSPP-E1 F 5′-TGCAAAAGTCCATGACAGTG-3′SEQ ID 128 NO:3 DSPP-E1 R 5′-TCAGTTGGTTCTGAGTAAAAAGGA-3′ SEQ ID NO:4DSPP-E2 F 5′-AAGTAATTTTGTGCTGTTCCTTT-3′ SEQ ID 149 NO:5 DSPP-E2 R5′-AACAAAGTGAAGAGGTTTTCT-3′ SEQ ID NO:6 DSPP-E3 F5′-AAGAACCTTTTCAATTGCTAGT-3′ SEQ ID 189 NO:7 DSPP-E3 R5′-TGGAGAAGTTAATGGAATGTAGCA-3′ SEQ ID NO:8 DSPP-E4 F5′-TGCAATTTGCTTTCCTTCAA-3′ SEQ ID 205 NO:9 DSPP-E4 R5′-CCTCTTCGTTTGCTAATGTGG-3′ SEQ ID NO:10 DSPP-E5 F5′-TCACAAGGTAGAAGGGAATG-3′ SEQ ID 226 NO:11 DSPP-E5 R5′-GTTTGTGGCTCCAGCATTGT-3′ SEQ ID NO:12 DSPP-E6 F5′-GGGACACAGGAAAAGCAGAA-3′ SEQ ID 243 NO:13 DSPP-E6 R5′-TGTTATTGCTTCCAGCTACTTGAG-3′ SEQ ID NO:14 DSPP-E7 F5′-CAATGAGGATGTCGCTGTTG-3′ SEQ ID 206 NO:15 DSPP-E7 R5′-TATCCAGGCCAGCATCTTCT-3′ SEQ ID NO:16 DSPP-E8 F5′-CACCTCAGATCAACAGCAAGAG-3′ SEQ ID 226 NO:17 DSPP-E8 R5′-TCTTCTTTCCCATGGTCCTG-3′ SEQ ID NO:18 DSPP-E9 F5′-ATGAAGAAGCAGGGAATGGA-3′ SEQ ID 232 NO:19 DSPP-E9 R5′-ATTCTTTGGCTGCCATTGTC-3′ SEQ ID NO:20 DSPP-E10 F5′-TGATGGAGACAAGACCTCCAA-3′ SEQ ID 205 NO:21 DSPP-E10 R5′-TGCCATTGAAAGAAATCAGC-3′ SEQ ID NO:22 DSPP-E11 F5′-TTCTTTCCTCCATCCTTCCA-3′ SEQ ID 194 NO:23 DSPP-E11 R5′-TTCTGATTTTTGGCCAGGTC-3′ SEQ ID NO:24 DSPP-E12 F5′-GGCAATGTCAAGACACAAGG-3′ SEQ ID 236 NO:25 DSPP-E12 R5′-TCTCCTCGGCTACTGCTGTT-3′ SEQ ID NO:26 DSPP-E13 F5′-TGCAAGGAGATGATCCCAAT-3′ SEQ ID 231 NO:27 DSPP-E13 R5′-TGTCATCATTCCCATTGTTACC-3′ SEQ ID NO:28 DSPP-E14 F5′-CAAAAGGAGCAGAAGATGATGA-3′ SEQ ID 243 NO:29 DSPP-E14 R5′-TGCTGTCACTGTCACTGCTG-3′ SEQ ID NO:30 DSPP-E15 F5′-GCAGTGATAGTAGTGACAGCAGTG-3′ SEQ ID 205 NO:31 DSPP-E15 R5′-TTGCTGCTGTCTGACTTGCT-3′ SEQ ID NO:32 DSPP-E16 F5′-CAAATCAGACAGTGGCAAAGG-3′ SEQ ID 508 NO:33 DSPP-E16 R5′-GCTCTCACTGCTATTGCTGCT-3′ SEQ ID NO:34 DSPP-E17 F5′-GCAAGTCAGACAGCAGCAAA-3′ SEQ ID 598 NO:35 DSPP-E17 R5′-CTGCTGTCGCTATCACTGCT-3′ SEQ ID NO:36 DSPP-E18 F5′-ATAGCAACGACAGCAGCAAT-3′ SEQ ID 583 NO:37 DSPP-E18 R5′-TCGCTGCTATTGCTATCACTG-3′ SEQ ID NO:38 DSPP-E19 F5′-GCAACAGCAGTGATAGTGACA-3′ SEQ ID 598 NO:39 DSPP-E19 R5′-CTGCTGTCGCTGCTTTCA-3′ SEQ ID NO:40 DSPP-E20 F5′-AGCAGCGACAGCAGTGATAT-3′ SEQ ID 500 NO:41 DSPP-E20 R5′-TTGTTACCGTTACCAGACTTGC-3′ SEQ ID NO:42 DSPP-E21 F5′-TGACAGCACATCTGACAGCA-3′ SEQ ID 261 NO:43 DSPP-E21 R5′-TCCCCCAGTTGTTTTTGTTT-3′ SEQ ID NO:44

[0080] PCR products were sequenced to determine the type and location ofmutations.

[0081] 1. The DNA fragments that showed a changed electrophoresispattern in SSCP analysis were amplified by standard PCR.

[0082] 2. PCR products were purified with Millipore spin column.

[0083] 3. Sequencing Reaction: (1) Reaction system Reaction mixture 2 ulPrimers (0.8 mM) 2 ul Purified PCR products 3 ul (2) Reactionconditions: 96° C. 30 sec 96° C. 30 sec 50° C.  5 sec 60° C.  4 min 60°C.  4 min

[0084] Total 35 cycles

[0085] (3) Precipitation of the Product of Sequencing Reaction

[0086] Add 9 volumes of 70% ethanol into the sequencing product,incubate at 4° C. for 3 minutes.

[0087] Centrifuge at 4° C. at 4000 rpm for 30 minutes.

[0088] Place the centrifuge tube upside down and continue to centrifugeuntil the speed reaches 1300 rpm at 4° C.

[0089] (4) Loading and Sequencing Samples

[0090] Add 2 ul Loading Dye buffer into precipitated products ofsequencing reaction.

[0091] Incubate at 90° C. for 2 minutes and place it on ice immediately.

[0092] Load samples into ABI PRISM automated DNA sequencer to sequence.

[0093] The sequencing results were shown in FIGS. 4A and 4B. In DGI-IIfamily, sequencing revealed a G1→T1 mutation at position 1 of Exon 3(position 3577 in SEQ ID NO:1). This mutation resulted in not only anamino acid change, but also a splicing site change that might cause theexpression of intron, termination of translation in advance or frameshifting (FIG. 4A). Therefore, the normal DSPP (or DSP) protein wasunable to be expressed.

[0094] In another DGI-II with deafness family, the mutation was a G1→A1mutation in position 1 of Intron 3 (position 3661 of SEQ ID NO:1). Thismutation was predicted to result in splicing site change which may causethe expression of intron, termination of translation in advance or frameshifting (FIG. 4B). Therefore, the normal DSPP (or DSP) protein wasunable to be expressed. Further, it might influence the translation ofsignal peptide so that DSPP could not be correctly localized.Surprisingly, this mutation caused the patient affected with both DGI-IIand deafness, suggesting that DSPP mutation was associated withdeafness. It is possible to diagnose deafness, especially DGI-II withdeafness, by detecting whether DSPP is normal or not.

[0095] Discussion

[0096] 1. Linkage and Haplotype Analysis

[0097] We used seven STR markers in 4q21 region to genotype DGI-II andDGI-II with deafness families. Linkage and haplotype construction showedthat the disease gene in DGI-II family was linked with 4q21 and themaximum LOD score was 8.38 at SPP1 locus (θ=0.00) (Table 1, FIG. 2) andthe disease gene in DGI-II with deafness was also linked with STRmarkers in 4q21 region and the maximum LOD score was 2.71 (0=0.00)(Table 2, FIG. 3).

[0098] 2. Mutation Screening of Candidate Genes and Confirmation bySequencing

[0099] We designed 22 primers overlapping the DSPP gene to screen formutations and identify mutations by sequencing. We found the diseasegene in DGI-II family was linked with the STR markers in 4q21 region,while the disease gene in DGI-II with deafness was also linked with STRmarkers in this region. These mutations were not observed in 100 normaland unaffected individuals. It suggests that these mutations should bethe cause of DGI-II disease.

[0100] DPP and DSP are two small polypeptides which have specificchemical properties and are cleaved from a single transcripts of DSPPgene. Both of them are expressed specifically in dental pulp tissue andmay also be expressed in cochleae. DSP is a Glu-, Ser- and Gly-richprotein with many phosphorylation sites, which are predicted to beinvolved in dentin mineralization. DPP affects mineralization in twoways. Low concentration of DPP protein is able to bind to interspace ofcollagen I and initiate formation of phosphorum apatite crystals, whilehigh concentration of DPP protein binds to the growing crystals, affectsthe size and form of crystals, and decreases the growth of crystals. Itis necessary to further study the mechanism that the mutations in DSPPgene cause dentinogenesis imperfecta and deafness.

[0101] All the documents cited herein are incorporated into theinvention as reference, as if each of them is individually incorporated.Further, it would be appreciated that, in the above teaching of theinvention, the skilled in the art could make certain changes ormodifications to the invention, and these equivalents would still bewithin the scope of the invention defined by the appended claims of thepresent application.

REFERENCES

[0102] 1. Witkop C J et al. Hereditary defects in enamel and dentin.Acta Genet 1957;7:236˜239

[0103] 2. Cetta G et al. Third international conference on osteogenesisimperfecta. Ann NY Avad Sci, 1998

[0104] 3. Takagi Y et al. Matrix protein difference between human normaland dentinogenesis imperfecta dentin. In the chemistry and biology ofmineralized connective tissues. Veis A, editor, New York:Elsevier/North-Holand. 1981

[0105] 4. Witkop C J, et al. Medical and dental findings in theBrandywine isolate. AL J Med Sci 1966;3:382˜403

[0106] 5. Bixler D, et al. Dentinogenesis imperfecta: genetic variationin a six-generation family. J. Dent. Res. 1968;48:1196˜1199

[0107] 6. Mikkelsen, M et al. Possible localization of Gc-system onchromosome 4. Loss of long arm 4 material associated with father-childincompatibility within the Gc-system. Hum. Hered. 1988;27: 105˜107

[0108] 7. Ball. S P, et al. Linkage between dentinogenesis imperfectaand Gc. Ann. Hum. Genet. 1982;46:35˜40

[0109] 8. Crall M G. Genetic marker study of dentinogenesis imperfecta.Proc Finn Dent Soc. 1992;88:285˜293

[0110] 9. Crodby A H, et al. Genetic mapping of dentinogenesisimperfecta type II Locus. Am. J. Hm. Genet. 1995;57:832˜839

[0111] 10. Aplin H. M, et al. Refinement of the dentinogenesisimperfecta type II locus to an interval of less than 2 centimorgans atchromosome 4q21 and the creation of a yeast artificial chromosome contigof the critical region. J. Dent. Res. 1999;78(6):1270˜1276

[0112]

1 44 1 8201 DNA Homo sapiens 1 attgtcatgc aaaagtccag gacagtgggccactttcagt cttcaaagag aaagataaga 60 aattctggat tttcaaaatc cttttgaagccttttaaggt aagatgaaat atccttttta 120 ctcagaacca actgattcat ttagaaagaactttgaattt caaagatgaa gccagtttga 180 ttttaagaag cgagtacccc ttaatgattagattgtatgc ttcctttttg acttgtcata 240 ttgatagtat gtataaaaga taacggacgattacgaccta aggaagagat agattgggaa 300 gaagaaagac ctcgtactga aaaattggccaactgaggtg gaaatttgac aattaactat 360 ctgggcactt tgattagttt tgataaaaaatgagataact cagatttcaa aaatccacct 420 tgggctttca aacaaggctt caattaggctttgcttttta gtattttatt acttactatt 480 acttattatt tattgtccca catgaaatgaaatttagcaa tcactaatga tgccaaatct 540 aattgctaaa tgaaatgaag ctaaatctcatttcattagt aacaataaat gaaataatct 600 gatggagctt cacaaattct gaagtctttgtttcatgctg aggtcacctg ggccattttt 660 attgtagtct tcgaagtcat tcacctgccttggaaacggt gataaccatc atggaattgt 720 tcaggagtgg agctgaaaga gagatgtagtggtcagattt ctgaactgta gctcagaaac 780 tggacacgta tcactctggc cttggctgcaggtacctttc cagtatgctg aggctcttcc 840 aaatcacagt gcagacgggc cttctgcagagctatgtaat gattaggctt gggactgcaa 900 agtacaggat aactgtggct tagtaaacagctggccttca acatctgtgc cccagagctc 960 tgcatgatac ttgtcctggt gtcacctcagcctcacttga atctatggca tttcagaagg 1020 agctctagct gttcttggct ttctgttgaacagctataag aatgagcact tttttccctc 1080 tcagtagctc tggaactgtg tcatctctcctgtgagaaaa cgccagtaat tctcatgaca 1140 gttgatattc agtgaagttt tattatattttcactaccac cattaaattc aatcaaagcc 1200 attttatgac atgcagcatt ataatctatacatctggtgg gagttcatga aataggagta 1260 aaactctcct ttctatcatt acttcaagaaatccaacttg caatataaat taattttttt 1320 actcacacag attataaaat gtctattccaacttatcaga aacatgtttt agaccatttc 1380 tgaatttgaa ttctaacagg gatgaagaatcatgatttta gaagtcccat aaaataattg 1440 ctatcattta ttcaaaaatt gcaaagtgcctgaagcaatg ctagatattg ctgatagtca 1500 taaatattta tcaacaacat tcagaaaacgtttttttctg tgctttgcat tggaatacaa 1560 taatcaccaa gacactctcc tgggcctcaggagcttacag gaaatcaggg caacacataa 1620 gtaactaggc aattttaaac agtgcaatgcgttaccagtg agacgtgcaa acttccttgg 1680 tataaaaagg aaagagatac caaataccctttgaagtggc gtcagagagg gcgtctcaga 1740 gataattcta ccaaacttca ggataatcctgaggtgcagg tgttgttatt attccaggtg 1800 gagggataat aaacctactt aaatttctcaagcttacaca gcaagtagca ggggtaacat 1860 ttgaacccag gtctctgaat acaaaccccgtattctttcc actagcgtag gctccctcat 1920 gttagtaatt tctttctctt aaagtctggtatagctcaat tctatagatt tggagtaagg 1980 atgacaagtg ttttaccttt gaagcacaatttcagcagaa ttagttagta cttgattaaa 2040 gctattcaga agagaaatag atgtttttacacccaagaat tgcagaagaa caaagttaca 2100 gctatgccct ttgtacctat tatggtgttttccttcattg gcacaggcag aaaaaaatct 2160 aggaagctac attagtgctg agcctggtgatgtccccata accacaccag gtatgttctg 2220 gaccatcgta tgtcttctcg tgttagatacatgcttcttg tccaggaaaa gggcaaatgc 2280 ttacacatca aaataatata gtactatgattttcccttta ctttataagt aattttgtgc 2340 tgttcctttt ttatacagcc attgattattattattccta aagaaaatga agataattac 2400 atatttttgc atttgggcag tagcatgggccattccagta agtatgcctt tcttagaaaa 2460 cctcttcact ttgttatctt ttttaacctaacattaatac aaaatgtagt gtgtgtgtgt 2520 gtgtgtgtgt gtgtgtgtgt gtgcatgtacatgtgtgtat atatgtgtgt gtgtatatat 2580 gtttccttaa ttttttttaa caggctgagtctaaacattt agatttgcac taagggcttt 2640 atgtgatatc tgtgaggttt caacaaaaccactccaattc atcgtctcat tcctctatag 2700 aaactcatat ctcgtctgaa ggattattattatttaaaac atttattcag attaatttac 2760 acttaatgcc cagaagtcat ggagactttgtccatctttg cttcatactc tgtgaatttc 2820 attctaatac gaacaaagtc tgtgctgtttaggaagtttc caagaaagaa taataagaaa 2880 aagtagattt tttttcaaca tataggagactaatttttca ctcagagtta ttatttatgt 2940 gctcactgtg gaaaatttgg aatatatgacgaaaaccaat aaaaaattga gaaaattcaa 3000 ccatttataa ttttactagc cagccatcatgtttaacatt ttcatatgct ttcataatac 3060 caaacatttg gtatttatgt agttgaaaatgttctcaagt atttcaaatg tgctcttgca 3120 gagcacagaa gtatactagc gtaatacttgattttgcttc tgtgcaggct ctggtcacgc 3180 ctcctgttct cttaagagtt ttcatcaggattacacttag agcgggtttg tgctagtgca 3240 agaggctttt tgtagagaaa caccagaggtctatcccctc gtctttctac aagactcttt 3300 ccttctacag ttgagataag tgggctgatctaacacgtcc ataaaattgg taataccaca 3360 gtgaaaaata tccatgtacc cagtttaaattctacacaag ccctgtaaga agccacttct 3420 cttttctatc tgattagatc atactttggcctttgtgtta aacctttctt cttcatggag 3480 ggaagaatat ttgtgtgtgt gtgtgtgtgtgtgcacgctc acacacatat tcacaaataa 3540 gaaccttttc aatagccagt attttctacttggcaggttc ctcaaagcaa accactggag 3600 agacatgtcg aaaaatccat gaatttgcatctcctagcaa gatcaaatgt gtcagtacag 3660 gtataggatg taatatattt cattttatttcctatttctg agttgctaca ttccattaac 3720 ttctccaaga ttgcaatttg ctttccttcaagatcattga cactcataat tgattgaatt 3780 gtttcttttt caggatgagt taaatgccagtggaaccatc aaagaaagtg gtgtcctggt 3840 gcatgaaggt gatagaggaa ggcaagagaatacccaagat ggtcacaagg gagaagggaa 3900 tggctctaag tgggcagaag taggagggaagagtttttct acatattcca cattagcaaa 3960 cgaagagggg aatattgagg gctggaatggggacacagga aaagcagaaa catatggtca 4020 tgatggaata catgggaaag aagaaaacatcacagcaaat ggcatccagg gacaagtaag 4080 catcattgac aatgctggag ccacaaacagaagcaacact aatggaaata ctgataagaa 4140 tacccaaaat ggggatgttg gcgatgcaggtcacaatgag gatgtcgctg ttgtccaaga 4200 agatggacct caagtagctg gaagcaataacagtacagac aatgaggatg aaataattga 4260 gaattcctgt agaaacgagg gtaatacaagtgaaataaca cctcagatca acagcaagag 4320 aaatgggact aaggaagctg aggtaacaccaggcactgga gaagatgctg gcctggataa 4380 ttccgatggg agtcctagtg ggaatggagcagatgaggat gaagacgagg gttctggtga 4440 tgatgaagat gaagaagcag ggaatggaaaagacagtagt aataacagca agggccagga 4500 gggccaggac catgggaaag aagatgatcatgatagtagc ataggtcaaa attcggatag 4560 taaagaatat tatgaccctg aaggcaaagaagatccccat aatgaagttg atggagacaa 4620 gacctccaag agtgaggaga attctgctggtattccagaa gacaatggca gccaaagaat 4680 agaggacacc cagaagctca accatagagaaagcaaacgc gtagaaaata gaatcaccaa 4740 agaatcagag acacatgctg ttgggaagagccaagataag gttagtttgt aaagctgatt 4800 tctttcaatg gcagtttaaa ttcttcccctccatctattg atgctagcac aaaaataaac 4860 catgacaagc atccatgtat ttttgtatccatattacttg actatttaag gaaatctaga 4920 gtccttacta gacttcgaga tagaacaactttaaacatct tacatttctg ataacttagt 4980 tataattcta gaaaagtctt atgtgaaatcatggatcccc atgtaattgt ttacaaaagt 5040 tcctactggg taggaatgtg gatgaatttttaaggaatct aagcaccagg atgctttcaa 5100 ttacagaata aagcacattt tcacaaataactgtgaagta ctagaaatgt aactcctatc 5160 cctatggcaa cttttcccag ttattcttcctcagatcaat gcaattttgc agcaaatatt 5220 cactagttaa tcattctttc ctccatccttccatagggaa tagaaatcaa gggtcccagc 5280 agtggcaaca gaaatattac caaagaagttgggaaaggca acgaaggtaa agaggataaa 5340 ggacaacatg gaatgatctt gggcaaaggcaatgtcaaga cacaaggaga ggttgtcaac 5400 atagaaggac ctggccaaaa atcagaaccaggaaataaag ttggacacag caatacaggt 5460 agtgacagca atagtgatgg atatgacagttatgattttg atgataagtc catgcaagga 5520 gatgatccca atagcagtga tgaatctaatggcaatgatg atgctaattc agaaagtgac 5580 aataacagca gtagccgagg agatgcttcttataactctg atgaatcaaa agataatggc 5640 aatggcagtg actcaaaagg agcagaagatgatgacagtg atagcacatc agacactaat 5700 aatagtgaca gtaatggcaa tggtaacaatgggaatgatg acaatgacaa atcagacagt 5760 ggcaaaggta aatcagatag cagtgacagtgatagtagtg atagcagcaa tagcagtgat 5820 agtagtgaca gcagtgacag tgacagcagtgatagcaaca gtagcagtga tagtgacagc 5880 agtgacagtg acagcagtga tagcagtgacagtgatagta gtgatagcag caatagcagt 5940 gacagtagtg acagcagtga tagcagtgacagtagtgata gtagtgacag cagtgacagc 6000 aagtcagaca gcagcaaatc agagagcgacagcagtgata gtgacagtaa gtcagacagc 6060 agtgacagca acagcagtga cagtagtgacaacagtgata gcagcgacag cagcaatagc 6120 agtaacagca gtgatagtag tgacagcagtgatagcagtg acagcagcag tagcagtgac 6180 agcagcagta gcagtgacag cagcaacagcagtgatagta gtgacagtag tgacagcagc 6240 aatagcagtg agagcagtga tagtagtgacagcagtgata gtgacagcag tgatagtagt 6300 gacagcagta atagtaacag cagcgatagtgacagcagca acagcagcga tagcagtgac 6360 agcagtgata gcagtgacag cagcaacagcagtgacagta gcgatagcag tgacagcagc 6420 aacagcagtg acagcagtga tagcagtgacagcagtgata gtagtgacag cagcaacagc 6480 agtgatagca acgacagcag caatagcagtgacagcagtg atagcagcaa cagcagtgat 6540 agcagcaaca gcagtgatag cagtgatagcagtgacagca gtgatagcga cagcagcaat 6600 agcagtgaca gcagtaatag tagtgacagcagcgatagca gcaacagcag tgatagcagc 6660 gacagcagcg atagcagtga cagcagtgatagcgacagca gcaatagaag tgacagtagt 6720 aatagtagtg acagcagcga tagcagtgacagcagcaaca gcagtgacag cagtgatagt 6780 agtgacagca gtgacagcaa cgaaagcagcaatagcagtg acagcagtga tagcagcaac 6840 agcagtgata gtgacagcag tgatagcagcaacagcagtg acagcagtga tagcagcaac 6900 agcagtgata gcagtgaaag cagtaatagtagtgacaaca gcaatagcag tgacagcagc 6960 aacagcagtg acagcagtga tagcagtgacagcagtaata gtagtgacag cagcaatagc 7020 ggtgacagca gcaacagcag tgacagcagtgatagcaata gcagcgacag cagtgacagc 7080 agcaacagca gcgatagcag tgacagcagtgatagcagtg acagcagtga cagcagtgat 7140 agcagcaaca gcagtgatag cagtgacagcagtgacagca gtgatagcag taatagtagt 7200 gacagcagca acagcagtga cagcagcgatagcagtgaca gcagcgatag cagtgacagc 7260 agtgacagca gcaatagcag tgacagcagtgacagcagcg acagcagtga tagcagtgac 7320 agcagtggca gcagcgacag cagtgatagcagtgacagca gtgatagcag cgatagcagt 7380 gacagcagcg acagcagtga cagcagtgacagcagtgaaa gcagcgacag cagcgatagc 7440 agcgacagca gtgacagcag cgacagcagtgacagcagcg atagcagcga cagcagcgac 7500 agcagcgata gcagtgacag cagcaatagcagtgatagca gcgacagcag tgatagcagt 7560 gacagcagcg acagcagcga tagcagcgacagcagtgata gtagtgatag cagtgacagc 7620 agtgacagca gcgacagcag tgacagcagcgacagcagtg acagcagcga cagcagtgac 7680 agcaatgaaa gcagcgacag cagtgacagcagcgatagca gtgacagcag caacagcagt 7740 gacagcagcg acagcagtga tagcagtgacagcacatctg acagcaatga tgagagtgac 7800 agccagagca agtctggtaa cggtaacaacaatggaagtg acagtgacag tgacagtgaa 7860 ggcagtgaca gtaaccactc aaccagtgatgattagaaca aaagaaaaac ccataagatt 7920 ccttttgtga aaagtttggt aatgggataggaaaaaaaga tttccaagaa agtaaagaaa 7980 ggggagaaat aaacataaga cgtatgtaaacaaaaacaac tgggggaatc aaatcaaaca 8040 gttggattca gaaccaagac ctaactcctgcagagacaga ctctgaatgc atgacctttg 8100 gtacatgcct gttaatattc atgttctgaaaatattttgt taaaagtgta aatctaaaca 8160 taaaagaaca attaaaatat tctttaatacttcacacaga a 8201 2 1253 PRT Homo sapiens 2 Met Lys Ile Ile Thr Tyr PheCys Ile Trp Ala Val Ala Trp Ala Ile 1 5 10 15 Pro Val Pro Gln Ser LysPro Leu Glu Arg His Val Glu Lys Ser Met 20 25 30 Asn Leu His Leu Leu AlaArg Ser Asn Val Ser Val Gln Asp Glu Leu 35 40 45 Asn Ala Ser Gly Thr IleLys Glu Ser Gly Val Leu Val His Glu Gly 50 55 60 Asp Arg Gly Arg Gln GluAsn Thr Gln Asp Gly His Lys Gly Glu Gly 65 70 75 80 Asn Gly Ser Lys TrpAla Glu Val Gly Gly Lys Ser Phe Ser Thr Tyr 85 90 95 Ser Thr Leu Ala AsnGlu Glu Gly Asn Ile Glu Gly Trp Asn Gly Asp 100 105 110 Thr Gly Lys AlaGlu Thr Tyr Gly His Asp Gly Ile His Gly Lys Glu 115 120 125 Glu Asn IleThr Ala Asn Gly Ile Gln Gly Gln Val Ser Ile Ile Asp 130 135 140 Asn AlaGly Ala Thr Asn Arg Ser Asn Thr Asn Gly Asn Thr Asp Lys 145 150 155 160Asn Thr Gln Asn Gly Asp Val Gly Asp Ala Gly His Asn Glu Asp Val 165 170175 Ala Val Val Gln Glu Asp Gly Pro Gln Val Ala Gly Ser Asn Asn Ser 180185 190 Thr Asp Asn Glu Asp Glu Ile Ile Glu Asn Ser Cys Arg Asn Glu Gly195 200 205 Asn Thr Ser Glu Ile Thr Pro Gln Ile Asn Ser Lys Arg Asn GlyThr 210 215 220 Lys Glu Ala Glu Val Thr Pro Gly Thr Gly Glu Asp Ala GlyLeu Asp 225 230 235 240 Asn Ser Asp Gly Ser Pro Ser Gly Asn Gly Ala AspGlu Asp Glu Asp 245 250 255 Glu Gly Ser Gly Asp Asp Glu Asp Glu Glu AlaGly Asn Gly Lys Asp 260 265 270 Ser Ser Asn Asn Ser Lys Gly Gln Glu GlyGln Asp His Gly Lys Glu 275 280 285 Asp Asp His Asp Ser Ser Ile Gly GlnAsn Ser Asp Ser Lys Glu Tyr 290 295 300 Tyr Asp Pro Glu Gly Lys Glu AspPro His Asn Glu Val Asp Gly Asp 305 310 315 320 Lys Thr Ser Lys Ser GluGlu Asn Ser Ala Gly Ile Pro Glu Asp Asn 325 330 335 Gly Ser Gln Arg IleGlu Asp Thr Gln Lys Leu Asn His Arg Glu Ser 340 345 350 Lys Arg Val GluAsn Arg Ile Thr Lys Glu Ser Glu Thr His Ala Val 355 360 365 Gly Lys SerGln Asp Lys Gly Ile Glu Ile Lys Gly Pro Ser Ser Gly 370 375 380 Asn ArgAsn Ile Thr Lys Glu Val Gly Lys Gly Asn Glu Gly Lys Glu 385 390 395 400Asp Lys Gly Gln His Gly Met Ile Leu Gly Lys Gly Asn Val Lys Thr 405 410415 Gln Gly Glu Val Val Asn Ile Glu Gly Pro Gly Gln Lys Ser Glu Pro 420425 430 Gly Asn Lys Val Gly His Ser Asn Thr Gly Ser Asp Ser Asn Ser Asp435 440 445 Gly Tyr Asp Ser Tyr Asp Phe Asp Asp Lys Ser Met Gln Gly AspAsp 450 455 460 Pro Asn Ser Ser Asp Glu Ser Asn Gly Asn Asp Asp Ala AsnSer Glu 465 470 475 480 Ser Asp Asn Asn Ser Ser Ser Arg Gly Asp Ala SerTyr Asn Ser Asp 485 490 495 Glu Ser Lys Asp Asn Gly Asn Gly Ser Asp SerLys Gly Ala Glu Asp 500 505 510 Asp Asp Ser Asp Ser Thr Ser Asp Thr AsnAsn Ser Asp Ser Asn Gly 515 520 525 Asn Gly Asn Asn Gly Asn Asp Asp AsnAsp Lys Ser Asp Ser Gly Lys 530 535 540 Gly Lys Ser Asp Ser Ser Asp SerAsp Ser Ser Asp Ser Ser Asn Ser 545 550 555 560 Ser Asp Ser Ser Asp SerSer Asp Ser Asp Ser Ser Asp Ser Asn Ser 565 570 575 Ser Ser Asp Ser AspSer Ser Asp Ser Asp Ser Ser Asp Ser Ser Asp 580 585 590 Ser Asp Ser SerAsp Ser Ser Asn Ser Ser Asp Ser Ser Asp Ser Ser 595 600 605 Asp Ser SerAsp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Lys Ser 610 615 620 Asp SerSer Lys Ser Glu Ser Asp Ser Ser Asp Ser Asp Ser Lys Ser 625 630 635 640Asp Ser Ser Asp Ser Asn Ser Ser Asp Ser Ser Asp Asn Ser Asp Ser 645 650655 Ser Asp Ser Ser Asn Ser Ser Asn Ser Ser Asp Ser Ser Asp Ser Ser 660665 670 Asp Ser Ser Asp Ser Ser Ser Ser Ser Asp Ser Ser Ser Ser Ser Asp675 680 685 Ser Ser Asn Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser AsnSer 690 695 700 Ser Glu Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Asp SerSer Asp 705 710 715 720 Ser Ser Asp Ser Ser Asn Ser Asn Ser Ser Asp SerAsp Ser Ser Asn 725 730 735 Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser SerAsp Ser Ser Asn Ser 740 745 750 Ser Asp Ser Ser Asp Ser Ser Asp Ser SerAsn Ser Ser Asp Ser Ser 755 760 765 Asp Ser Ser Asp Ser Ser Asp Ser SerAsp Ser Ser Asn Ser Ser Asp 770 775 780 Ser Asn Asp Ser Ser Asn Ser SerAsp Ser Ser Asp Ser Ser Asn Ser 785 790 795 800 Ser Asp Ser Ser Asn SerSer Asp Ser Ser Asp Ser Ser Asp Ser Ser 805 810 815 Asp Ser Asp Ser SerAsn Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser 820 825 830 Ser Asp Ser SerAsn Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser 835 840 845 Asp Ser SerAsp Ser Asp Ser Ser Asn Arg Ser Asp Ser Ser Asn Ser 850 855 860 Ser AspSer Ser Asp Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser 865 870 875 880Asp Ser Ser Asp Ser Ser Asp Ser Asn Glu Ser Ser Asn Ser Ser Asp 885 890895 Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Asp Ser Ser Asp Ser Ser 900905 910 Asn Ser Ser Asp Ser Ser Asp Ser Ser Asn Ser Ser Asp Ser Ser Glu915 920 925 Ser Ser Asn Ser Ser Asp Asn Ser Asn Ser Ser Asp Ser Ser AsnSer 930 935 940 Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asn Ser Ser AspSer Ser 945 950 955 960 Asn Ser Gly Asp Ser Ser Asn Ser Ser Asp Ser SerAsp Ser Asn Ser 965 970 975 Ser Asp Ser Ser Asp Ser Ser Asn Ser Ser AspSer Ser Asp Ser Ser 980 985 990 Asp Ser Ser Asp Ser Ser Asp Ser Ser AspSer Ser Asn Ser Ser Asp 995 1000 1005 Ser Ser Asp Ser Ser Asp Ser SerAsp Ser Ser Asn Ser Ser Asp 1010 1015 1020 Ser Ser Asn Ser Ser Asp SerSer Asp Ser Ser Asp Ser Ser Asp 1025 1030 1035 Ser Ser Asp Ser Ser AspSer Ser Asn Ser Ser Asp Ser Ser Asp 1040 1045 1050 Ser Ser Asp Ser SerAsp Ser Ser Asp Ser Ser Gly Ser Ser Asp 1055 1060 1065 Ser Ser Asp SerSer Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp 1070 1075 1080 Ser Ser AspSer Ser Asp Ser Ser Asp Ser Ser Glu Ser Ser Asp 1085 1090 1095 Ser SerAsp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp 1100 1105 1110 SerSer Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp 1115 1120 1125Ser Ser Asn Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp 1130 11351140 Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp 11451150 1155 Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asp1160 1165 1170 Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Asn Glu Ser SerAsp 1175 1180 1185 Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Ser Asn SerSer Asp 1190 1195 1200 Ser Ser Asp Ser Ser Asp Ser Ser Asp Ser Thr SerAsp Ser Asn 1205 1210 1215 Asp Glu Ser Asp Ser Gln Ser Lys Ser Gly AsnGly Asn Asn Asn 1220 1225 1230 Gly Ser Asp Ser Asp Ser Asp Ser Glu GlySer Asp Ser Asn His 1235 1240 1245 Ser Thr Ser Asp Asp 1250 3 20 DNAArtificial Sequence Primer 3 tgcaaaagtc catgacagtg 20 4 24 DNAArtificial Sequence Primer 4 tcagttggtt ctgagtaaaa agga 24 5 23 DNAArtificial Sequence Primer 5 aagtaatttt gtgctgttcc ttt 23 6 21 DNAArtificial Sequence Primer 6 aacaaagtga agaggttttc t 21 7 22 DNAArtificial Sequence Primer 7 aagaaccttt tcaattgcta gt 22 8 24 DNAArtificial Sequence Primer 8 tggagaagtt aatggaatgt agca 24 9 20 DNAArtificial Sequence Primer 9 tgcaatttgc tttccttcaa 20 10 21 DNAArtificial Sequence Primer 10 cctcttcgtt tgctaatgtg g 21 11 20 DNAArtificial Sequence Primer 11 tcacaaggta gaagggaatg 20 12 20 DNAArtificial Sequence Primer 12 gtttgtggct ccagcattgt 20 13 20 DNAArtificial Sequence Primer 13 gggacacagg aaaagcagaa 20 14 24 DNAArtificial Sequence Primer 14 tgttattgct tccagctact tgag 24 15 20 DNAArtificial Sequence Primer 15 caatgaggat gtcgctgttg 20 16 20 DNAArtificial Sequence Primer 16 tatccaggcc agcatcttct 20 17 22 DNAArtificial Sequence Primer 17 cacctcagat caacagcaag ag 22 18 20 DNAArtificial Sequence Primer 18 tcttctttcc catggtcctg 20 19 20 DNAArtificial Sequence Primer 19 atgaagaagc agggaatgga 20 20 20 DNAArtificial Sequence Primer 20 attctttggc tgccattgtc 20 21 21 DNAArtificial Sequence Primer 21 tgatggagac aagacctcca a 21 22 20 DNAArtificial Sequence Primer 22 tgccattgaa agaaatcagc 20 23 20 DNAArtificial Sequence Primer 23 ttctttcctc catccttcca 20 24 20 DNAArtificial Sequence Primer 24 ttctgatttt tggccaggtc 20 25 20 DNAArtificial Sequence Primer 25 ggcaatgtca agacacaagg 20 26 20 DNAArtificial Sequence Primer 26 tctcctcggc tactgctgtt 20 27 20 DNAArtificial Sequence Primer 27 tgcaaggaga tgatcccaat 20 28 22 DNAArtificial Sequence Primer 28 tgtcatcatt cccattgtta cc 22 29 22 DNAArtificial Sequence Primer 29 caaaaggagc agaagatgat ga 22 30 20 DNAArtificial Sequence Primer 30 tgctgtcact gtcactgctg 20 31 24 DNAArtificial Sequence Primer 31 gcagtgatag tagtgacagc agtg 24 32 20 DNAArtificial Sequence Primer 32 ttgctgctgt ctgacttgct 20 33 21 DNAArtificial Sequence Primer 33 caaatcagac agtggcaaag g 21 34 21 DNAArtificial Sequence Primer 34 gctctcactg ctattgctgc t 21 35 20 DNAArtificial Sequence Primer 35 gcaagtcaga cagcagcaaa 20 36 20 DNAArtificial Sequence Primer 36 ctgctgtcgc tatcactgct 20 37 20 DNAArtificial Sequence Primer 37 atagcaacga cagcagcaat 20 38 21 DNAArtificial Sequence Primer 38 tcgctgctat tgctatcact g 21 39 21 DNAArtificial Sequence Primer 39 gcaacagcag tgatagtgac a 21 40 18 DNAArtificial Sequence Primer 40 ctgctgtcgc tgctttca 18 41 20 DNAArtificial Sequence Primer 41 agcagcgaca gcagtgatat 20 42 22 DNAArtificial Sequence Primer 42 ttgttaccgt taccagactt gc 22 43 20 DNAArtificial Sequence Primer 43 tgacagcaca tctgacagca 20 44 20 DNAArtificial Sequence Primer 44 tcccccagtt gtttttgttt 20

What is claimed is:
 1. A method for determining the susceptibility ofDGI-II and/or DGI-II with deafness in a subject comprising the steps of:detecting the DSPP gene, transcript and/or protein in said subject andcomparing it with the normal DSPP gene, transcript and/or protein todetermine whether there is any difference, wherein said differenceindicates that the possibility of suffering DGI-II and/or DGI-II withdeafness in said subject is higher than the normal population.
 2. Themethod of claim 1 wherein the DSPP gene or transcript is detected, andcompared with the normal DSPP nucleotide sequence to determine thedifference.
 3. The method of claim 2 wherein said difference is selectedfrom the group consisting of: in position 1 of Exon 3, G1→T1; inposition 1 of Intron 3, G1→A1.
 4. A method for treating DGI-II and/orDGI-II with deafness comprising the step of administrating a safe andeffective amount of normal DSPP and/or DSP protein to the patient inneed of said treatment.
 5. The method of claim 4 wherein the DSPP and/orDSP protein is administrated topically to periodontal tissues.
 6. Apharmaceutical composition comprising a safe and effective amount ofDSPP and/or DSP protein and a pharmaceutically acceptable carrier. 7.The pharmaceutical composition of claim 6 which is injection.
 8. A kitfor detecting DGI-II and/or DGI-II with deafness comprising the primerswhich specifically amplify the DSPP gene or transcript.
 9. The kit ofclaim 8 which further comprises a probe that binds to the site ofmutation.
 10. The kit of claim 9, wherein the mutation is selected fromthe group consisting of: in position 1 of Exon 3, G1→T1; in position 1of Intron 3, G1→A1.